Ligand based solution assay for low concentration analytes

ABSTRACT

The present invention provides an assay for an analyte in solution. The assay relies, in part, on associating two catalytic activities in close proximity to each other (e.g., by conjugating each enzyme to a ligand that binds the analyte) and providing substrates that produce a colored product only when both activities are bound to the same molecule in solution.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. provisional patent applicationsNo. 60/240,442, filed Oct. 14, 2000, and No. 60/244,065, filed Oct. 28,2000. The contents of each are incorporated herein.

FIELD OF THE INVENTION

This invention relates to calorimetric detection of analytes in a liquidsample, and finds application in the fields of biology, medicalanalysis, and analytical chemistry.

BACKGROUND

This section discusses a variety of methods for detection of compoundsby the appearance of color. However, the citation of a reference orconcept in this section should not be construed as an indication thatthe reference or concept is prior art to the present invention.

Detecting and measuring color is a convenient method for measuring theamount of a substance in solution. If the substance to be detected,i.e., the “analyte”, does not have an inherent color, a color may beproduced, as surrogate for the substance, by a variety of chemical,enzymatic or immunochemical methods. This well known art is practiced,for example, in both research and clinical laboratories of biologicaland health care fields. The principle of colorimetry is the constantloss of light in passage through a solution by absorbance of light intothe colored compound. A molecular species absorbs the same amount oflight in proportion to its concentration at the same wavelength everytime it is measured. Light lost as it passes through a solution isdetermined by the concentration of the absorbing molecules and thelength of the light path. By knowing the length of the light path andthe light loss and the volume of the solution, it is possible tocalculate the amount, or mass, of a substance. In practice, thecalculation is frequently replaced by a standard curve represented bythe same reaction on a series of known amounts of the same substancethat have been processed by the same reactions to produce color. Inalternative methods, the amount of light lost by scattering can bedetermined.

Ligand Assay

The sensitivity of a colorimetric test is defined as the limit amountthat may be detected reliably using the method. One way to increasesensitivity (i.e., lower the limit amount to be detected), is to use anamplifier method for producing color. Amplifiers of special interest tolife science assay design are catalysts, and especially the class ofbiological catalysts known as enzymes. Color reactions for the detectionof enzymes or for the detection of the substrates on which enzymesoperate as catalysts are well known. Sensitivity may be improved furtherby attaching enzymes to molecules that recognize the analyte, usuallyreferred to as ligands.

ELISA

One standard assay for detecting and quantifying an analyte in asolution is the Enzyme Linked Immunosorbent Assay, or ELISA. However,this assay can be difficult to carry out and expensive. In this assay,after the enzyme linked ligand is joined to all analyte present in theassay, the excess enzyme linked ligand that is not attached to analytemust be eliminated from the solution or it produces unwantedamplification signal. The standard assay design includes a series ofsteps as follows:

One example of a standard container for such assays is a 96 well plate,so called because it has a matrix of 12 by 8 wells of standard size in astandard size frame. Manufacturers may treat such plates so as to permitstrong attachment of certain molecular species to the well walls.

Adding a solution containing ligand to each well on the plate at 4° C.permits attachment and retains activity of ligand. This is an“overnight” procedure.

Plates are then washed to eliminate any ligand not firmly attached tothe well wall. There are three sequential washes.

Plates are most often used immediately because the ligands so attachedare not stable to storage. Sample or standard analyte solutions orblanks containing reagent only are added to individual wells. Theanalyte species, which forms the ligand pair, attaches to the ligand onthe well wall.

After some period of incubation the plates are again washed to eliminatethe remaining sample solution. There are three sequential washes

Enzyme-conjugated ligand is then added to each well. Usually this ligandhas specificity for another recognition site on the analyte molecule.

The plate is again washed to eliminate excess enzyme-conjugated ligandremaining in solution. There are three sequential washes. At this pointthe amount of enzyme attached to the wall of each well is determined bythe amount of analyte also attached to the well wall.

Reagents are then added to test for the presence of the enzyme, and thecolor so produced relates to the amount of analyte added to each well.

Measurement is made in a photometer designed for reading the plates,called a plate reader.

It is apparent that ELISA is a tedious, time-consuming assay with manynecessary steps. A manufacturer may perform the initial preparation ofplates. Such manufactured plates are expensive. For example, one assayplate for performing 96 tests, may cost $650. In use, the assay usingsuch a plate still takes approximately 5 hours to complete.

Channeling

One improvement over ELISA methods is known as “channeling” described inGibbons et al., Methods of Enzymology 136:93. The principle ofchanneling is to form small, specialized particulates during the assay.The particulates permit attachment of ligands with two separate enzymes.The enzymes act in coordination, such that the product of one enzymeacts as substrate for the next. Only enzymes attached to the particlespermit channeling. Enzyme not attached to particles do not produce colorreaction product. Although this is a theoretical improvement over ELISA,there are considerations in formation of such specialized particles,which make this design impractical.

Other Solution Assays

Other solution assays using amplifiers are known. For example, U.S. Pat.No. 3,975,237 discloses a solution assay typically for small molecules.The assay principle is an inhibition of enzyme activity by use of alarge molecule receptor, for example an antibody to the small molecularweight analyte, as competition to a small molecular version of the sameanalyte molecule. Methods of preparing conjugates of enzymes withanalytes, and the sensitivity of assays of this nature are described.Another solution assay is described by Kricka and Ji, 1994, ClinicalChemistry 40:1828 -30. This assay uses small molecule aryl boronic acidsto enhance enzymatic luminescence. In a similar assay described in U.S.Pat. Nos. 5,843,666 and 5,306,621, the chemiluminescence is furtherenhanced by small molecule phenols. The method uses a binding partnerlabeled with a hydrolytic enzyme to produce a phenolic enhancer in closeproximity to a peroxidase labeled specific binding partner. Themechanism of the enhancement is not known. This is a luminescence assaythat uses expensive equipment that is not available to large numbers oflaboratories, and has limited sensitivity.

Histochemistry

In the fields of histochemistry and cytochemistry, color contrast intissues or cells is produced for purpose of microscopic examination ordetection. Ligands that recognize tissue components and conjugated toenzymes are used as amplifiers that may then produce color withappropriate reagents. For visual examination it is possible to provideseveral colors of reaction product for several individual analytes withseveral different enzyme-conjugated ligands. This method is acceptableas long as the different analytes are located in different cells ortissue components. However, if the two analytes are present in the samelocation the resulting colors are additive, producing a new color whichcannot be interpreted by microscopy. Another way to resolve two colorsin one location is to use fluorescent markers. However fluorescencemicroscopy is much more expensive, and the automatic detection offluorescence requires longer integration times, thus making automationof two color image detection impractical.

Color Photographic Development

Exposure of color film to light produces activated silver granules inthe film. Light of different colors activates silver particles indifferent layers of the color film. During development with a commonreagent, silver grains are reduced and thereby oxidize the commondeveloper. The oxidized developer is captured in the layer chemicallycombines with color couplers. It is only the product of couplingoxidized developer and color coupler that produces color. Each layer hasat least one coupler producing a color reaction product specific forthat layer. Scavenger molecules, sometimes called “white couplers,”prevent diffusion of developer from one layer to another. It isconsidered an advantage of the reaction of scavengers and coloredcouplers with oxidized developers if the reaction product is retained inthe layer where it is formed. This is accomplished by designing orselecting couplers that are insoluble in the developer solvent bothbefore and after coupling takes place. Some scavengers and some colorcouplers are designed with attachment to immobilized polymers. There areactive regions on color coupler molecules and white couplers whichenhance coupling to oxidized or activated photographic developers.

The chemical structure of the color couplers is highly similar to thereaction product of histochemical color producing compounds. Indeed thehistochemical and cytochemical substrates are often the same asphotographic color couplers with addition of a protective group on theactive site. The protective group is hydrolyzed from the active regionby action of the enzyme of interest. The chemical structure ofphotographic color developers is also very similar to substrates used inhistochemistry of peroxidase reactions. The oxidized reaction product isalso similar in histochemistry and in color photographic developers. Inboth chemical reactions the oxidation potential is also approximatelythe same. Use of photographic developers in detection of reactionproducts of hydrolytic enzymes was suggested by, for example Ornstein,1959, Histochemistry and Cytochemistry, 7: 231 and Ornstein, 1974,Histochemistry and Cytochemistry 22:453-69, both incorporated byreference herein.

In the photographic industry it is well known that certain couplers formcolor more efficiently than others. By this is meant that they requireless or more oxidized developer to form color. Since the amount ofoxidized developer is determined by the number of sensitized silverions, the effect is to require more sensitized silver ions for somecouplers than for others. The more efficient couplers are known as2-equivalent couplers, while the less efficient couplers are known as4-equivalent couplers.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an assay for analytes in solution. Theassay relies, in part, on associating two catalytic activities in closeproximity to each other to produce a detectable product. The inventiontakes advantage of the proximity of two catalytic activities, forexample enzyme activities, to limit production of color, while the sameenzymes not in proximity produce only minor background color. Thepresent invention makes use of compounds analogous to protected colorcouplers and analogs to photographic color developers to produce colorthat is limited to regions in the solution where an oxidizing enzyme anda hydrolytic enzyme are in proximal location. In one embodiment of theinvention white couplers are used as scavengers to inhibit colorproduction in the solution where the enzymes are not in proximity.

The present invention has advantages over existing methods for detectinganalytes in solution, such as ELISA. For example, the present assay canbe carried out in multi-well (e.g., 96-well) plates without any washsteps to remove non-bound ligand, non-analyte molecules in the sample,and non-bound enzyme conjugated ligand. Without any attachment ofcapture reagent, analyte or detection reagent to the well wall, thepresent invention permits completion of a test within 1 to 1.5 hours.The results can be read using colorimetry plate readers, widelyavailable clinical and research laboratories. Thus, the assay of thepresent invention is more rapid, has fewer steps, and is more economicalthan existing methods.

In one aspect, the invention provides a method for detecting an analytein solution comprising (a) combining (i) a solution to be assayed forthe presence or amount of the analyte; (ii) a first ligand capable ofbinding the analyte, wherein said first ligand is directly or indirectlybound to a first enzyme capable of cleaving a first substrate to producea colorless first product, wherein said first enzyme is a hydrolase;(iii) a second ligand that binds the analyte, wherein the binding of thefirst ligand to the analyte does not interfere with the binding of thesecond ligand, and wherein the second ligand is directly or indirectlybound to a second enzyme capable of oxidizing a second substrate toproduce a colorless second product, wherein said second enzyme is anoxidase; (iv) said first substrate; and, (v) said second substrate;whereby the hydrolase cleaves the first substrate to product the firstproduct and the oxidase oxidizes that second substrate to produce thesecond product, wherein the first product and the second productchemically combine to produce a detectable reaction product, saiddetectable reaction product being a colored reaction product; (b)detecting the production of the colored reaction product; (c) relatingthe production of the colored reaction product with the presence ofanalyte in the solution. The method can further comprise combining acompound that is a scavenger for the first reaction product or thesecond reaction product in step (a). The scavenger can be3-amino-1-(2,4,6-trichlorophenyl)-2-pyrazolin-5-one or acetoacetamide.The first substrate can be a compound that comprises a benzene ring ornaphthalene structure with one active hydroxyl group, e.g., 1-naphtholphosphate or phenyl phosphate. The second substrate can be N,N-dimethylparaphenylene diamine; N,N-diethyl paraphenylene diamine; N-phenylparaphenylene diamine;N′-ethyl-N′ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine; 4 amino antipyrene; or N,N-dimethylamino benzidine. In variousembodiments, the first ligand is a first antibody that specificallybinds the analyte and second ligand is a second antibody thatspecifically bind the analyte, the hydrolase is a phosphatase, anesterases, a galactosidase, a lipase, a glucuronidase, an amidase, apeptidase, or a sulphatase. In an embodiment, for example the hydrolaseis alkaline phosphatase and the oxidase is horseradish peroxidase. In anembodiment, the first substrate is naphthyl phosphate or phenylphosphate and the second substrate isN′-ethyl-N′ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine.In some embodiments, at least one of the first and second ligand is anantibody or a lectin.

In a related aspect, the invention provides a method for detecting ananalyte in solution comprising (a) combining (i) a solution to beassayed for the presence or amount of the analyte, wherein said analytehas an oxidase activity capable of acting on a first substrate toproduce a colorless first product; (ii) a ligand capable of binding theanalyte, wherein said ligand is directly or indirectly bound to a firstenzyme capable of cleaving a second substrate to produce a colorlesssecond product, wherein said first enzyme is a hydrolase; (iii) saidfirst substrate; and, (v) said second substrate; whereby the hydrolasecleaves the second substrate to product the second product and theoxidase oxidizes the first substrate to produce the first product,wherein the first product and the second product chemically combine toproduce a detectable reaction product, said detectable reaction productbeing a colored reaction product; (b) detecting the production of thecolored reaction product; (c) relating the production of the coloredreaction product with the presence of analyte in the solution. In anembodiment, the analyte has a pseudoperoxidase activity. In anembodiment, the analyte is glycated hemoglobin. In an embodiment, thesolution comprises non glycated hemoglobin and the glycated portion ofhemoglobin to be compared to total hemoglobin. In various embodiments,the ligand is an organic boronic acid compound directly or indirectlyconjugated to a hydrolase. Further, the hydrolase can be alkalinephosphatase; the method can include combining a compound that is ascavenger for the first reaction product in step (a); the firstsubstrate is selected from the group N,N-dimethyl paraphenylene diamine;N,N-diethyl paraphenylene diamine; N-phenyl paraphenylene diamine;N′-ethyl-N′ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine; 4 amino antipyrene; and N,N-dimethylamino benzidine, the secondsubstate is naphthyl phosphate or phenyl phosphate, and the scavanger is3-amino-1-(2,4,6-trichlorophenyl)-2-pyrazolin-5-one or acetoacetamide.

In a related aspect, the invention provides a method for detecting ananalyte in solution comprising (a) combining (i) a solution to beassayed for the presence or amount of the analyte, wherein said analytehas a hydrolase activity capable of acting on a first substrate toproduce a colorless first product; (ii) a ligand capable of binding theanalyte, wherein said ligand is directly or indirectly bound to a firstenzyme capable of cleaving a second substrate to produce a colorlesssecond product, wherein said first enzyme is a oxidase; (iii) said firstsubstrate; and, (v) said second substrate; whereby the hydrolase cleavesthe first substrate to produce the first product and the oxidaseoxidizes the second substrate to produce the second product, wherein thefirst product and the second product chemically combine to produce adetectable reaction product, said detectable reaction product being acolored reaction product; (b) detecting the production of the coloredreaction product; (c) relating the production of the colored reactionproduct with the presence of analyte in the solution.

Stated differently, in various aspects and embodiment, the inventionprovides (1) A quantitative or qualitative colorimetric solution assayfor analytes comprising: providing an analyte in solution; providing afirst ligand to the analyte; providing a second ligand to the analyte;providing a catalytic activity for the first ligand to the analyte;providing a different catalytic activity for the second ligand to theanalyte; providing a reagent for the first catalytic activity devised togive a first colorless reaction product; providing a reagent for thesecond catalytic activity devised to give a second colorless reactionproduct; devising conditions where the further reaction of the firstreaction product and the second reaction product produces a coloredthird reaction product only when the first ligand and the second ligandare attached to the same analyte molecule; detecting the third reactionproduct by the amount of color produced and relating the detected colorto the analyte in solution. In various embodiments: a reagent acting asa scavenger for the first or second reaction product is provided; thecatalyst attached to the first ligand is an enzyme, such as an oxidase(e.g., horseradish peroxidase); the catalyst attached to the secondligand is an hydrolase enzyme (e.g., alkaline phosphatase); the secondligand is attached to a different epitope or attachment than the firstligand; first catalyst is horseradish peroxidase, the second catalyst isalkaline phosphatase, the first reagent is an oxidizable developer andthe second reagent is naphthyl phosphate or phenyl phosphate; theoxidisable developer isN′-ethyl-N′ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine;the reaction comprises scavenger (e.g.,3-amino-1-(2,4,6-trichlorophenyl)-2-pyrazolin-5-one or acetoacetamide).

In various embodiment, at least one ligand is an antibody; or at leastone ligand is a lectin; or at least one of the ligands is a moleculewith more general affinity properties (e.g., a boronic acid compound)

In one aspect, the invention provides: a quantitative or qualitativecolorimetric solution assay for analytes comprising: providing ananalyte with a first catalytic activity in solution; providing a ligandto the analyte; providing a second catalytic activity for the ligand tothe analyte; providing a reagent for the first catalytic activitydevised to give a first colorless reaction product; providing a reagentfor the second catalytic activity devised to give a second colorlessreaction product; devising conditions where the further reaction of thefirst reaction product and the second reaction product produces acolored third reaction product only when the ligand is attached to theanalyte; detecting the third reaction product by the amount of colorproduced and relating the detected color to the analyte in solution.

In various embodiments, a scavenger for the first or second reactionproduct is included, the catalyst analyte is an enzyme or pseudoenzyme;the enzyme is an oxidase (e.g., peroxidase or pseudoperoxidase) thecatalyst attached to the ligand is a hydrolase enzyme (e.g., alkalinephosphatase); the ligand is attached to a different epitope orattachment than the enzyme or active site of the analyte; the firstcatalyst is a peroxidase, the second catalyst is alkaline phosphatase,the first reagent is an oxidizable developer and the second reagent isnaphthyl phosphate or phenyl phosphate.

In various embodiments, the first catalyst is glycated hemoglobin; thereis present in the analyte sample a quantity of non glycated hemoglobinand the result to be determined is the determination of the glycatedportion of hemoglobin compared to total hemoglobin; the oxidizabledeveloper isN′-ethyl-N′ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine;a scavenger reagent is included (e.g.,3-amino-1-(2,4,6-trichlorophenyl)-2-pyrazolin-5-one or acetoacetamide);the ligand is an antibody; the ligand is a lectin; the ligand is amolecule with more general affinity properties (e.g. a boronic acidcompound, attached to alkaline phosphatase.

In a related aspect, the invention provides a kit for solution assayscomprising a first antibody to an analyte conjugated to a first enzymewith peroxidase activity, a second antibody to the same analyteconjugated to a second enzyme with alkaline phosphatase activity, asource of hydrogen peroxide, an oxidizable developer, a phenol likesubstrate for alkaline phosphatase and a colorless coupler to use asscavenger. In an embodiment, the oxidizable developer isN′-ethyl-N′ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine,the substrate is phenyl phosphate or naphthyl phosphate and thescavenger is acetoacetamide. The invention also provides a kit fordetecting the proportion of glycated to total hemoglobin comprising awell plate suitable for measurement in a plate reader, an enzyme withalkaline phosphatase activity coupled with a boronic acid, a source ofhydrogen peroxide, an oxidizable developer, a phenol like substrate foralkaline phosphatase and a colorless coupler to use as scavenger. In anembodiment, the oxidizable developer isN′-ethyl-N′ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine,the substrate is naphthyl phosphate and the scavenger is acetoacetamide.

DESCRIPTION OF THE FIGURES

FIG. 1 provides an exemplary reaction of the type that can be used inthe present invention.

FIG. 2 provides results of a model solution assay, as described inExample 1.

FIG. 3 provides results of a model solution assay, as described inExample 2.

FIG. 4 provides results of a model solution assay, as described inExample 3.

FIG. 5 provides results of an assay for GM-CSF, as described in Example4.

DETAILED DESCRIPTION OF THE INVENTION

Overview

The present invention provides a method for detecting an analyte insolution. In one embodiment, a hydrolase enzyme activity and an oxidaseenzyme activity are brought into close proximity by joining both enzymesto a single analyte molecule and appropriate substrates provided suchthat the hydrolase acts on one substrate to produce a non-coloredsoluble product, the oxidase acts on a second substrate to produce anon-colored soluble product, and, provided the two products are producedin close proximity, they chemically combine to produce a soluble coloredproduct that can be detected. The appearance of the colored product iscorrelated with the quantity of the analyte in the sample, e.g., byusing a standard curve. In a preferred embodiment, one or morescavenger(s) is present during the reaction that reacts with uncoloredproduct(s). As used herein, the terms “chemically combine,” refers tothe formation of a covalent chemical linkage between two reactionproducts, resulting in a third product that is detectable anddistinguishable from the two reaction products.

A summary of one embodiment of the method is provided in FIG. 1, forillustration and not limitation. A variety of other embodiments aredisclosed herein. In addition, a variety of histochemical andcytochemical principles and reagents are disclosed in U.S. patentapplication Ser. No. 09/411,352, published Apr. 12, 2001 as WO 01/25476,which can be applied in the present method, provided that combinationsof the reagents are selected to remain soluble. This can be accomplishedby selecting or modifying reagents to increase solubility, or,alternatively, by including a solubilizing agent (e.g., 1-10% ethanol,diethylene glycol, and the like) in the reaction mixture. In the presentmethod, proximity of two enzymatic activities at the molecular level isrequired, which means that the two primary reaction products must reacttogether to form a final reaction product in molecular proximity.

Analyte

As used herein, the term “analyte” refers to the compound in solution tobe detected using the assay of the invention. The analyte can be any ofa variety of compounds or macromolecular complexes in solution such as,without limitation, a polynucleotide, an antigen, a hapten, an antibody,a viral particle, and the like. In one embodiment, as described below,the analyte has an enzymatic activity. The term “solution,” as usedherein, refers to an aquous solution which can be a buffered solution, ahomogenate (e.g., cell homogenate), a body fluid (e.g., plasma, urine,cerebrospinal fluid), an extract or partially purified compound, or thelike, suspected of containing the target analyte. The methods of thepresent invention are most useful for detection of analytes at lowconcentration (e.g., typically less than 10 micrograms/ml, often lessthan 1 microgram/ml).

Ligands and Ligand Conjugates

The present invention employs binding molecules, also called “ligands,”that bind an analyte molecule in solution. Typically the binding isnon-covalent. In one embodiment of the invention, two ligands are usedwhich bind to the same analyte molecule. For example, in one embodiment,two antibodies (ligands) bind two different epitopes of a protein ormacromolecular complex. That is, the antibodies are paired antibodies tothe same analyte, but with a different specific binding site (epitope)on the same analyte. When used in the context of ELISA tests, suchantibody pairs are often referred to as a “capture antibody” and a“detection antibody.”

Suitable antibodies for use in the invention include monoclonalantibodies, binding fragments (e.g. Fab fragments), single chainantibodies, and the like. In addition to antibodies, suitable ligandsinclude lectins (which specifically bind carbohydrates) and otherbinding molecules described herein or well known in the art (e.g.,biotin, avidin, protein A). An inhibitor analog of a substrate thatbinds to an enzyme active site is another example of a ligand. As usedherein, no particular biological function, other than selective bindingto the analyte, is implied by use of the term ligand. Certain small(i.e., molecular weight less than 1000) molecules have specificity for aclass of structures and are useful in analysis. For exampleboronate-containing compounds have an affinity for hydroxyl groups ontwo or more adjacent carbon atoms. Boronate conjugated enzymes, forexample aminophenyl boronic acid conjugated to alkaline phosphatase canbe used as ligands for the detection of carbohydrates (e.g., providedthe other component of the paired enzyme conjugate has sufficientspecificity, as described below).

The ligand(s) is directly or indirectly bound, or coupled to, an enzymeor other catalyst. As used in this context, “direct” binding means thatthe catalyst (enzyme) is covalently bound to the ligand (e.g.,antibody). Indirect binding refers to any of a variety of art-knownmeans for covalently or non-covalently associating an enzyme with abinding molecule: examples include use of an enzyme labeled secondaryantibody that binds a primary (anti-analyte) antibody, biotin-avidinmediated binding, and the like. Stated differently, ligands forconjugation with the enzymes include antibodies that are specific forthe analytes to be measured. The well-known ligand pairs such as theavidin-biotin pair may be applied as secondary linkages to provide amore versatile reagent. For example a biotinylated nucleotide may beused in a hybridization assay where temperature of the procedure mayinactivate a directly linked enzyme. Following hybridization the enzymemay be linked through avidin to the hybridized molecule.

Enzyme-ligand Conjugates

In certain embodiments in which two ligands are used, each of the two isconjugated to an enzyme (i.e., protein catalyst) with a differentsubstrate specificity (i.e., a different enzyme). In one embodiment, oneligand is conjugated to an oxidase and a second ligand is conjugated toa hydrolase. It will be apparent that when the analyte of interest isone half of a ligand pair, and the other half is attached to an enzymeamplifier, then the analyte is indirectly also joined to the enzymewhich is used as an amplifier.

Oxidase Enzymes

Oxidases are enzymes that catalyze the oxidation of a substrate. Any ofa variety of oxidase enzymes are suitable for use in the invention.Usually, the oxidase can use a photographic developer as substrate.Photographic developers, as is well known, are soluble organic compoundscharacterized by susceptibility to oxidation by sensitized silver ions,but not by nonsensitized silver ions, and the ability to couple with asecond compound having an active site. Virtually all photographicdevelopers are N,N-alkyl substituted para phenylenediamines. Suitableoxidases include peroxidases (e.g., horseradish peroxidase,myeloperoxidase), galactose oxidases, cytochrome oxidases, monoamineoxidases, pseudoperoxidases, and others. In some embodiments, asdescribed herein, the analyte itself may have an oxidase activity (i.e.,an endogenous oxidase activity). Examples of such analytes withendogenous oxidase activity include the pseudoperoxidase activity of thehemoglobin and the myoglobin molecules.

Oxidase Substrates

Generally, any oxidase substrate which is converted by an oxidase (e.g.,peroxidase) to a product capable of coupling with a color coupler(described herein below) to produce a soluble reaction product may beused in the methods disclosed herein. Suitable substrates include thosewith the same molecular structures as well-known photographicdevelopers. Exemplary substrates (sometimes referred to as “developers,”“photographic developers,” or “color developers”) include N,N-dimethylparaphenylene diamine; N,N-diethyl paraphenylene diamine; N-phenylparaphenylene diamine;N′-ethyl-N′ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine; 4 amino antipyrene; N,N-dimethylamino benzidine. A larger listof fifty such photographic developers may be found in Bent et al., 1951,J. Am. Chem. Soc. 73:3100-24, incorporated by reference herein in itsentirety. Additional oxidases and substrates useful in the presentinvention are described in copending U.S. patent application Ser. No.09/0411,352, published Apr. 12, 2001 as WO 01/25476, both incorporatedby reference herein in its entirety for all purposes. Developerperoxidase substrates may have chemical groups added to the phenyl ringwithout detriment to their reaction, and some groups are helpful indesign of certain assays. For example a methyl group ortho to theprimary amine is known in the photographic industry to preventautopolymerization of the oxidized developer. The dialkyl amine of N,Ndialkyl substituted phenylene diamine may also be beneficially modified.For example, addition of a sulphonamido group augments solubility of thefinal reaction product.

Autopolymerization may be used as a design feature which is useful forsome assays. For example, autopolymerization effectively prevents anoxidized developer molecule from coupling with a color coupler if thecoupling is not accomplished immediately after the oxidation. Thecomparable speeds of coupling and autopolymerization, two competitivereactions, will determine the diffusion distance from originalproduction at which color formation may occur. However, if the reactionis prolonged, there is a tendency for autopolymerized developer toprecipitate and thereby change the optical characteristics of thesolution to that of a suspension. Therefore a developer compound withouta methyl group ortho to the primary amine is more useful in rapidreactions, while a compound with such a methyl group is used in designof an assay that develops slowly. The latter assay is also more likelyto require a scavenger, as described below.

A preferred developer isN-ethyl-N-ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine.Thus, in one embodiment, N,N dialkyl substituted phenylenediamine isused as substrate in combination with a peroxidase.

It will be appreciated that, depending on the enzyme, certain cofactorsmay be required for the oxidation reaction. For example, when aperoxidase enzyme is used, hydrogen peroxide is also used as acosubstrate for the oxidation reaction. When certain oxidases are used,cytochrome C may be required as a cofactor.

Hydrolase Enzymes

Hydrolases are enzymes that catalyze a hydrolysis reaction, e.g.,capable of removing a protecting moiety from a substrate (color coupler)to produce a reactive product (color coupler). Exemplary hydrolasesinclude phosphatases (e.g., alkaline pbosphatase), esterases (e.g.,cholinesterases, carboxyl esterase), galactosidases (e.g., alphanaphtholbetagalactosidase), lipases, glucuronidases, amidase, peptidases, andsulphatases. These are all well known in the art (see, e.g., U.S. Pat.No. 3,975,237, incorporated herein by reference). Preferred hydrolyticenzymes are those which are more active at alkaline pH, because theseare the conditions preferred by the photographic chemical compounds. Aparticularly preferred enzyme, for reasons of economy and commercialavailability, is alkaline phosphatase.

Hydrolase Substrates

Substrates for hydrolases include esters, amides, peptides, ethers, orany chemical compound having an enzymatically hydrolyzable covalentbond. The enzyme catalyzed hydrolysis reaction results in a hydroxyl oran amine compound as one product (and free phosphate, acetate, etc., asa second product). Hydrolase enzyme substrated used in the presentinvention are those converted to a color coupler by the action of theenzyme by removal of a blocking group. The presence of the blockinggroup prevents the chemical combination of the substrate with theoxidized developer. Thus, typically the substrate has the formula:“R—B”, where “R” is a color coupler moiety “—B” is a blocking group suchas a phosphate, a sulfate, an acetate or a butyrate, or the like, linkedto the color coupler moiety by an enzyme-cleavable (e.g.,hydrolase-cleavable) linkage such as an ester (including a phosphateester or a sulfate ester) linkage or an amide linkage.

The preferred substrate for the hydrolase is a substituted (protected)phenol or naphthol compound (e.g., a preferred substrate for alkalinephosphatase is a phosphate-substituted phenol or naphthol compound. Thesubstrates generally preferred for histochemistry and cytochemistryapplications are not preferred substrates for the present inventionbecause they are designed to precipitate in the tissues or cellslocalizing the enzyme activity. The preferred substrates for the presentinvention are smaller molecules, such as 1-naphthol phosphate, or phenylphosphate, which remain in solution after hydrolysis and also aftercoupling with the preferred developer. Substitutions on these simplenaphthol and benzene compounds may enhance the reactivity or formationof the preferred color without detriment to their use. For examplesubstituting a chlorine group in the position para to the phosphategroup permits faster coupling and a more efficient reaction becausechlorine acts as a leaving group in the position to which the oxidizeddeveloper couples. These more efficient substrates are comparable to2-equivalent couplers known in the photographic industry. (In thephotographic industry it is well known that certain couplers form colormore efficiently than others, i.e., they require less or more oxidizeddeveloper to form color. Since the amount of oxidized developer isdetermined by the number of sensitized silver ions, the effect is torequire more sensitized silver ions for some couplers than for others.The more efficient couplers are known as 2-equivalent couplers, whilethe less efficient couplers are known as 4-equivalent couplers.) Thechoice of a 2-equivalent or 4-equivalent coupler type of substrate isdetermined by the relative activity of the two enzymes in any specificassay described in this invention. For example, twice as much activeperoxidase has to be in proximity of the hydrolase when using a4-equivalent coupler than when using a 2-equivalent coupler. Avidin is atetravalent ligand, and when using Avidin-conjugated hydrolase therewill be multiple liganded oxidases in proximity and a 4-equivalentcoupler is acceptable. When the enzymes in ligand related proximity arein 1:1 proportion, a 2-equivalent coupler is efficient.

Substrates for hydrolytic enzymes usually contain the enzyme specificcomponent adjacent to the benzene or naphthalene ring. In variousembodiments, the substrate comprises a substituted phenol or naphtholcompound, i.e., a compound that comprises a benzene ring or naphthalenestructure with one active hydroxyl group protected by theenzyme-specific substrate composition, and having at least one carbon inthe ring in a position ortho or para to the active hydroxyl group wherea substitution reaction may take place. Such ortho or para position maybe covered by hydrogen or by a halogen or other group which readilyleaves the ring structure during a substitution reaction. The substrateactive component is a specific structure varying with each enzyme. Thesubstrate for sulphatase may be 1-naphthyl sulfate. The substrate foreach caspase is a four amino acid peptide, which varies with thespecific caspase, but always has an aspartate residue adjacent to thenaphthol or phenol. After action of enzyme on the substrate, the benzeneor naphthalene will have a free (unprotected) hydroxyl group at the sitewhere the specific substrate component was attached. The free hydroxylactivates the ortho, or preferentially, the para position on the ringand permits coupling in these positions. If a halogen, carboxyl, orother efficient leaving group, is substituted in the ortho or paraposition then coupling in that position replaces the leaving groupduring the coupling reaction and the reaction is more efficient than inan unsubstituted naphthalene or benzene ring. Conversely a nitro orother strongly binding substituent in the ortho or para positions on thephenol will prevent coupling in that position but may permit coupling inthe alternate para or ortho position. For example, para-nitrophenylphosphate, after cleaving the phosphate becomes para-nitrophenol whichis not an efficient coupler with oxidized developers. The “leavinggroup” truly leaves during a coupling reaction, and the resulting colorcompound has the same color whether there was a leaving group present ornot. For example 4-chloro-1-naphthol or 1-naphthol coupled toN,N-diethyl-para-phenylene diamine have identical light absorptionspectra, but coupling (measured by development of color) of the 4-chlorois at least twice as fast.

The naphthol AS substrates which are preferable in histochemistrybecause their unprotected reaction products are poorly soluble inaqueous solutions are not preferred substrates for the solution assay.Just the opposite is preferred, in that a solubilizing group such as asulphonic acid on the opposing ring to the hydroxyl group, for example8-hydroxy-1-naphthalene sulphonic acid produces a highly colored, verysoluble, coupled compound with a very sharp absorption peak in the farblue region of the spectrum. An equivalent 8-phospho-1-naphthalenesulphonic acid would not couple and there would be no color. Thefollowing table provides hydrolase substrates. In the table, exemplaryhydrolases are provided along with blocking groups that can beconjugated to chromogenic moieties (i.e., color couplers). Part A showsthe hydrolase and group recognized by the enzyme, which is linked by ahydrolysable bond (“—”) to a chromogenic group such as those shown inPart B of the table.

TABLE 1 Hydrolase Substrates for Solution Assays Substrate specificcomponent is linked to the chromogenic coupler through a covalent bondindicated by two dashes (--) in the following tables. Part A: Hydrolasesand Substrate specific components (Examples) 1. Caspases Caspase 1Ac—Tyr—Val—Ala—Asp-- Caspase 3 Ac—Asp—Glu—Val—Asp-- Caspase 4Ac—Leu—Glu—Val—Asp-- Caspase 5 Ac—Trp—Glu—His—Asp-- Caspase 6Ac—Val—Glu—Ile—Asp-- Caspase 9 Ac—Leu—Glu—His—Asp-- 2. GlycosidasesGlucosidases Glucose-alpha- (alpha and beta) Glucose-beta-Galactosidases Galactose-alpha- Galactose-beta- Glucosaminidase N-Acetylglucosamine-- Glucuronidase Glucuronic acid-- 3. Peptidases andproteinases Collagenase 1 HO—Darg—Gln—Gly—Ala—Ill—Gly—Gln—Pro-- ElastaseIII Pyr—Pro—Val-- (Pyr = pyroglutamyl) Trypsin Benzoyl DL Arginine- 4.Esterases Various Acetate-- Chloroacetate-- Butyrate- 5. Inorganicesterases Phosphatase HO(OO)PO-- Sulphatase HO(OO)SO— Part B:Chromogenic (coupler) components (--all linking through hydroxyl oramine groups) Hydroxybenzine (Phenol) 4-chloro-1-Hydroxybenzine(4-Chlorophenol) 2-chloro-1-hydroxybenzine (2(chlorophenol) Aminobenzine(anilin) 4-chloro-1-aminobenzine (4-chloro-1-aminobenzine)2-chloro-1-aminobenzine (2-chloro-1-aminobenzine) 1-Naphthol 2-Naphthol4-chloro-1-naphthol 8-hydroxy-naphthaline-1-sulphonic acid 4-nitrophenol2-chloro-4-nitro-phenol 2-chloro-4-nitro-1 naphthol5-nitro-8-hydroxy-naphthalene-1 sulphonic acid

Scavengers

In the present context, a scavenger is a compound with the chemicalcoupling characteristics of the photographic color couplers, i.e., willreact with a photographic developer, but with minimal or no colorcontribution to the solution in the wavelengths where the measurementsare to be made. Scavengers are effective competitors to the colorcoupling reaction in the same solution, except in the region where bothenzymes are in close proximity, where the concentration of the freecolor coupler is expected to be much higher. It is possible for a singlechemical compound to act as a scavenger under some assay conditions andas a color coupler under different assay conditions.8-hydroxy-naphthalene sulphonic acid is an example. If it is the colorcoupler, then the scavenger with which it is paired will have either nocolor at all or will absorb in the far red part of the spectrum.

Exemplary scavnger compounds include 8-hydroxy naphthalene sulphonicacid; o-acetoacetanisidide; acetoacetamide; ethyl acetoacetate;1-(4-hydroxyphenyl)-1H-tetrazole-5-thiol;3-(2,4,6-trichlorophenyl)-aminopyrazoline-5-one; Tyramine;3-Hydroxytyramine; 4-aminoantipyrine; 4-hydroxyantipyrine;4-(hydroxymethyl)-4-methyl-1-phenyl-3-pyrazolidinone;1-phenyl-3-pyrazolidinone; with acetoacetamide;3-(2,4,6-trichlorophenyl)-aminopyrazoline-5-one; and 3-Hydroxytyraminepreferred.

Scavengers need not be completely color free after coupling, but shouldhave a narrow spectrum of color that does not interfere when the colorof measurement is carefully chosen. For example, a naphthol orphenol-containing substrate always gives a blue color on coupling andabsorbs best in the 650 to 690 nM region of the spectrum. A scavengerthat absorbs at 410 to 450 nm, but not above 600 nm, when coupled withthe same developer, provides a good matched pair.

Useful scavengers include chemical analogs of white couplers used in thephotographic arts, and include soluble triazole and tetrazole compounds.Other white couplers are listed in, for example U.S. Pat. No. 6,013,428,incorporated by reference herein, and these compounds, or analogs ofthem, may be useful as scavengers to prevent unwanted color formation.However the requirements of photographic white couplers are not exactlythe same as requirements for the present solution assay. The preferredscavengers for solution assay according to the present invention arecompounds that remain soluble after coupling with the developer. Incontrast, a number of the white couplers listed in U.S. Pat. No.6,013,428 are designed to prevent diffusion from one layer of film toanother layer after coupling. They have large hydrocarbon side chainsthat have no other purpose than to make them less soluble in water.Scavengers of this sort generally are not used in the present invention.The best criteria for an effective scavenger in the present inventioninclude water solubility before and after coupling, long term stabilityin aqueous solutions, no color before or after coupling in the spectralregion of measurement, and no interaction with the enzymes used in theassay.

Analyte Molecule with Enzymatic Activity

In an alternative embodiment of the invention, the analyte molecule hasthe additional reactive property of one of the enzymes described above.In this embodiment, only one enzyme-conjugated ligand is required forthe preferred reaction. When the enzyme bound ligand reagent attaches tothe specific analyte it effectively forms an enzyme pair. One example ofthis is the detection of fetal hemoglobin with an antibody to fetalhemoglobin conjugated to alkaline phosphatase. Fetal hemoglobin acts asa pseudoperoxidase and the proximity of the hydrolytic enzyme to theanalyte (having oxidase activity) satisfies the conditions hereindescribed.

In one embodiment, a hydrolase, e.g., alkaline phosphatase, conjugatedto a boronic acid moiety, e.g., aminophenyl boronic acid, is used as aligand. This compound attaches to glycated hemoglobin as well as otherglycated proteins. However only glycated hemoglobin has thepseudoperoxidase activity to provide the proximity of reaction productsto satisfy the conditions of the present invention. One useful assayexample is detection of glycated hemoglobin in the presence of otherglycated proteins and of non glycated hemoglobin by use of boronateconjugate of alkaline phosphatase because only the glycated hemoglobinacts as ligand-pair for boronate and acts as an enzyme as well. Otherboronic acid moieties, e.g., butyl boronic acid, can also be used.

In some embodiments, as described herein, the analyte may have ahydrolytic enzyme activity. Analytes with hydrolytic activity includecomponents of the blood clotting cascade of enzymes, the enzymesinvolved in programmed cell death (caspases), and phosphatases that maybe circulating in blood plasma normally or during a disease process.

Enzyme with Substrate Binding Activity

In a particular alternative embodiment of the invention, the analyte isan enzyme. Certain enzymes also strongly attach their substrates totheir active sites. After operating on the substrate, the product isreleased. Inhibitor compounds similar to the substrate can besynthesized which attach even more strongly to the active site of theenzyme, and do not permit subsequent release. If such an enzyme is theanalyte, then inhibitor with the characteristics described can beconsidered a ligand. Such a ligand may be conjugated directly orindirectly to an enzyme used as reagent in the present invention. Oneexample of such a ligand, described by N. A. Thornberry and Y. Lazebnik,1998, Science, 281: 1312 is: Biotin-X-Val-Ala-Asp(OMe)—CH₂F (where X isa linking group). This compound is the biotinylated derivative ofCaspase Inhibitor I. It may be used in the present invention togetherwith, for example, Avidin conjugated alkaline phosphatase to link thehydrolase enzyme indirectly to the Caspase analyte. A complete caspaseassay design also would contain an antibody to the specific caspaseconjugated to horseradish peroxidase, and also supplying the substratesboth the phosphatase and peroxidase, as previously described.

Exemplary Assay Format

The assay of the invention can be carried out in a variety of formats.The following description is for illustration and not for limitation. Inone embodiment, a multi-well plate, such as a 96-well plate, is used.The two enzyme-ligand conjugates used for detection are mixed inapproximately equal amounts and, typically, in amounts calculated to beapproximately equal to, or in small excess of, the highest concentrationof the analyte to be detected. While the 96 well plate is kept at aconstant, cold temperature, the solution of enzyme-ligand mix isdistributed in wells of the 96 well plate. In a series of at least 6wells there is distributed a series of dilutions of the analyte of knownconcentration. Preferably another 6 wells are used as duplicate for theanalyte dilutions of known concentration. In at least one, butpreferably at least 3 wells no analyte is placed but instead the samebuffer, used for the analyte dilutions, is used in the same volume ofbuffer. The unknown analyte is then distributed into yet other wells.Preferably the unknown analyte is used in duplicate wells for eachsample. When all samples and blanks have been placed in the wells,incubation is continued for time to allow ligand binding to occur, e.g.,typically at least 15 minutes and up to 12 hours, but preferably for 30minutes, at the same cold temperature.

On completion of incubation, the developing solution is added. Bufferconcentration of all components including the developing solution is inthe range of 10 to 200 mM. Preferably the buffer concentration is in therange of 20 mM to 100 mM, and is most preferably 50 mM.

For alkaline phosphatase paired with horseradish peroxidase, the pH ofbuffer is in the range of about 7.4 to about 10.4. More preferably thepH is in the range of about 8.0 to about 9.3 and most preferably isabout 8.3. Concentration of hydrogen peroxide is in the range of 0.01 to0.1%. More preferably the range of hydrogen peroxide concentration is0.02 to 0.09% and most preferably hydrogen peroxide is used as 0.03% ofthe final solution. Substrates are used in concentration range of 50 μMto 10 mM. More preferably the substrate concentrations are used as 100μM to 5 mM and most preferably in concentration of 1.5 mM. Colordevelopment is measured with a plate reader.

When the substrate pairs are the preferredN′-ethyl-N′ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine,and phenyl phosphate the preferred wavelengths of measurement are 694 nmand 405 nm. When a white coupler with slight yellow color is used, e.g.,acetoacetamide, the preferred wavelengths of measurement are 694 and 450nm in the plate reader. Following measurement at the appropriatewavelength pair, the result of measurement of the unknown is compared tothe result of measurement of the reference analyte and the concentrationof the unknown is determined by standard curve fitting methods.

In the preferred embodiment of the invention sample is added to apre-mixed solution of the two enzyme conjugated ligands in wells in a 96well plate. Sample is permitted to incubate with enzyme conjugatedligands for approximately one half hour, before adding the developersolution. The aqueous developer solution in the preferred embodimentcontains a photographic developer or analogous compound, a phosphatesubstituted phenol analog of a protected color coupler, a white coupleror analog of same which remains soluble during the reaction time,hydrogen peroxide and a buffer to control the acidity of the solution.Color develops steadily in the solution containing all these componentsplus the enzyme pair. The preferred time of reading the color in aconventional plate reader is at approximately 1 hour. But readings canbe made as early as 15 minutes and as late as 4 hours depending on thespecific application and reagents.

Kits

The reagents useful for practicing the methods of the present inventioncan be provided in kit form. In one embodiment, the kit comprises acontainer including separate vials of one or more of. (1) a hydrolasesubstrate that can be hydrolized to produce a color coupler (such as aspecific color coupler listed herein); (2) an oxidizable developer (suchas a specific oxidizable developer listed herein) (3) a hydrolaseconjugated to antibody (4) an oxidase (such as a specific oxidase listedherein) conjugated to an antibody (5) a scavenger (such as a specificscavenger listed herein) (6) a buffer. Typically at least two or atleast three of the above-listed reagents are included.

EXAMPLES

All examples presented here are model experiments with dilution seriesof an analyte. The subject analyte of the experiment was dissolved in anaqueous solution (in the same buffer used in the remainder of theassay). A specified volume of a specified concentration of the analytewas placed in a first well of a row of 12 wells on a 96 well plate. Anequal volume of buffer was placed in all 12 wells of the same row. Afterthe analyte and buffer in the first well were thoroughly mixed, onevolume equivalent was removed and transferred to the next well, where itwas again mixed thoroughly. This process of double dilution wascontinued for all except the last or 12^(th) well, which was left as ablank. The excess volume from well 11 was discarded, so that all wellshave an equal volume, while the concentration of analyte in the row of12 wells varies from a maximum in the first well (well number 1) to azero level in the last well (well number 12).

Example 1 Dilution Series of Avidin (Analyte) with Biotin-conjugatedEnzymes

Conditions of assay: Buffer: 50 mM Tris hydroxymethyl aminomethane,adjusted to pH 8.3 with HCl. The buffer also contains sodium chloride,potassium chloride and magnesium chloride (i.e., 200 mL of finalsolution contained 0.605 g of Tris hydroxymethyl aminomethane, 1.15 gNaCl, 0.15 g KCl and 0.1 g MgCl₂. All volumes placed in the wells are100 μL.

Solutions of Avidin, biotinylated alkaline phosphatase (AP-B) andbiotinylated horseradish peroxidase (HRP-B), all from Sigma Chemicals,St. Louis Mo., were prepared as 0.05 mg/mL stock. A further dilution ofAvidin was with 1.5 mL of buffer and 75 μL of Avidin stock solution. Theenzyme conjugates were diluted by adding 50 μL of HRP-B and 8.5 μL ofAP-B stock solutions to 10 mL of buffer. These first dilutions of stockwere kept in an ice bath or in the refrigerator until used and are notretained as dilutions from day to day.

Developer solution was made up as 12 mL of buffer containing 300 μL of3% hydrogen peroxide, 300 μL of a 50 mM stock solution ofN′-ethyl-N′ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine,150 μL of 3-amino-1-(2,4,6-trichlorophenyl)-2-pyrazolin-5-one, asscavenger, and 300 μL of 1-naphthol phosphate. All reagent stocksolutions were prepared as 50 mM stocks.

100 μL of dilute Avidin solution was added to the first well andserially diluted as described above. An exact replica of this serialdilution was performed in the next set of 12 wells. 100 μL of dilutedenzyme mix was added immediately after completion of the serialdilution. The prepared plate in which all wells contained Avidin (atvarious concentrations) as well as HRP-B and AP-B at constantconcentration, was kept on a cold surface for 30 minutes. Packaged andfrozen blue ice covered by several layers of wet paper towel provides aconvenient cold surface. After 30 minute incubation, 100 μL of adeveloper solution is added, and a measurement is taken at time ofmixing. In the experimental stage, measurements are taken at 5 minuteintervals to confirm the optimum design time. The measurements reportedfor this experiment are taken at 1 hour after adding developer. Thewavelength of measurement is absorbance at 450 nm subtracted fromabsorbance at 694 nm (OD 694-450 nm). The wavelengths of lightabsorbance measurement were determined by taking preliminary lightabsorption spectra separately of solutions of the reaction product ofoxidized developer with the color coupler and the reaction product ofthe oxidized developer with scavenger. A filter setting on the platereader that incorporates maximum absorbance of the developer-couplerproduct and minimum absorbance of the developer-scavenger product waschosen as the main wavelength. A filter setting where both compoundsgive minimum readings is chosen as the wavelength to be subtracted fromthe main wavelength.

Optical density measurements were transferred to a spreadsheet and theresults are plotted. The results are presented in FIG. 2. It can be seenthat the dilutions are in satisfactory linear order from the original120 nanograms in the first well to the 9^(th) serial dilution. This isequivalent to slightly less than 0.5 nanograms or 500 picograms ofAvidin. Since the molecular weight of Avidin is 60 kD the sensitivity ofthe measurement is in the order of 0.01 picomoles of the analyte.

In this experiment the proportion of AP-B to HRP-B is approximately 1:4because 1-naphthyl phosphate produces 1-naphthol as color coupler. Underconditions of the assay 1-naphthol acts as a 4-equivalent couplertherefore more peroxidase than phosphatase activity is required.

If this experiment is repeated but alkaline phosphatase without biotinis substituted for AP-B, at the same activity as would be in the AP-B,all the wells read at approximately the background level of the aboveexperiment. I.e. all readings are as if there is no Avidin in thesolution, although the Avidin dilutions are as in the describedexperiment. Surprisingly, the methods of the present invention result inan efficient, quantitative colored third reaction product only when thefirst ligand and the second ligand are attached to the same analytemolecule.

Example 2 Phenyl Phosphate Substrate

This experiment has identical conditions to Example 1, except that AP-Band HRP-B were both used at 50 μL of stock in 10 mL of buffer, and thesubstrate for AP was phenyl phosphate. An additional set of wells istreated identically except that polyethylene glycol 600 was present inthe developer at 4% concentration. The wavelengths of measurement were694-405 nm, because the phenyl coupler has a different peak absorptionand has no absorbance in the region of 400 nm. Results are presented inFIG. 3. The negative values are due to some absorbance of the3-amino-1-(2,4,6-trichlorophenyl)-2-pyrazolin-5-one, acting as ascavenger, in the 405 nm region. In preliminary experiments it was foundthat four times the activity of alkaline phosphatase is required forphenyl phosphate than for naphthyl phosphate. Because AP acts lessefficiently on phenyl phosphate than on 1-naphthyl phosphate, theproportions of enzyme were adjusted accordingly for this experiment.Avidin is a four valent molecule. That means that four biotinylatedmolecules can attach to one Avidin. As a result it is a good modelanalyte for determining the chemical interactions and for optimizingproportions of enzyme, and other reagents.

Results with polyethylene glycol 600 demonstrate that substances otherthan the main reactants may interfere with the reaction. In this casethere is an enhancement of sensitivity, possible due to volume exclusioneffects of PEG. Use of a standard curve accounts for such effects.

Example 3 Use of a Colorless Scavenger with Phenyl Phosphate Substrate

The conditions of experiment 2 were again repeated, but with use ofphenyl phosphate, and with equal amount volumes of stock HRP-B and AP-B.In addition, the scavenger used in experiments 1 and 2 was replaced with12, 24 or 48 μL acetoacetamide in 2 mL of developer solution. Readingswere taken at 650-405 nm. Results provide a regression curve similar toFIG. 3, except that there are no negative values in the regressionbecause the scavenger forms a colorless coupled compound with oxidizeddeveloper. See FIG. 4. Note that excess scavenger can compete with themain color reaction. I.e., all else being equal, using twice as muchscavenger will reduce the color to about half, because the scavenger iscompeting everywhere. The optimum concentration of scavenger can beadjusted to eliminate background color with “no sample blanks” and yetproduces maximum color with sample present.

These model experiments are used to demonstrate that conditions may bevaried in an experimental model in order to suite requirements of aspecific assay. The experimental conditions are sufficiently versatileto provide for the needs of assays of biological analytes.

Example 4 Dilution Series of a Low Concentration Biological Analyte

Granulocyte Monocyte colony stimulating factor (GM-CSF), antibody toGM-CSF conjugated with HRP, and a second antibody against GM-CSFconjugated with AP were obtained from R & D Systems, Minneapolis MN. TheGM-CSF and antibodies were prepared as stock solutions (0.05 mg/mL) inTris buffer with sodium, potassium and magnesium chlorides adjusted topH 7.4. 200 mL of the final buffer solution contains 0.605 g of Trishydroxymethyl aminomethane, 1.15 g NaCl, 0.15 g KCl and 0.1 g MgCl₂.

A dilution series of GM-CSF was prepared as described supra, with theconcentration ranging from 40 ng/100 μL in well number 1, and subsequent1:1 dilutions in buffer in wells 2-11 (with well 12 left as a blank).Antibody is supplied to the final mix as 50 μL of each stock in 10 mLbuffer. (All biologicals are made up as 0.05 mg/mL as stock. Thedilution for this experiment was 50 μL in 10 mL, i.e., a 200 folddilution from stock before adding 50 μL to each well. This is equivalentto 6.25 ng/well.)

The developer was as in experiment 3, with acetoacetamide as scavengerand phenyl phosphate as substrate, except that the pH of the developerwas adjusted to 10. When the developer was added to each well the finalpH is 8.3. Thus the ligand incubation was at pH 7.4 and the colordevelopment was at pH 8.3. The results shown in FIG. 5 were afterincubation overnight. However, similar results were obtained withshorter, room temperature incubations (e.g., 1-2 hours). Colordevelopment was for one hour. The results again show a dose responseregression when plotted graphically.

This experiment has sensitivity to approximately 0.04 nanograms which isequivalent to 0.025 picomoles because the molecular mass of GM-CSF isapproximately 14,000 D.

Example 5 Determine Percent Glycated Hemoglobin in a Solution Assay

In this example the buffer used was different from Examples 1-4, becauseTris buffer acts as competitor for the ligand, aminophenyl boronic acid.The analyte, glycated hemoglobin, and a control protein, non-glycatedhemoglobin were prepared by column chromatography. The column wascharged with aminophenyl boronic acid agarose purchased from Isolab. Thecolumn was washed with dilute hydrochloric acid and then equilibratedwith 100 mM glycylglycine (GG) buffer at pH 9.0. Whole blood washemolized with GG buffer containing Triton-X 100 (as the hemolizingagent). The lysate was layered on the column and left to stand for 5minutes, during which time the glycated hemoglobin in the blood sampleattaches to the boronated agarose. The first fraction, the non-glycatedhemoglobin, was then eluted with GG buffer at pH 9.0. The peak of thisfraction was isolated and preserved for further processing. The secondfraction was then eluted with Tris buffer pH 8.6, containing 50 mMsorbitol. The peak of this fraction was also collected for furtherprocessing. The column was then renewed with dilute hydrochloric acidand was equilibrated again with GG buffer. The first fraction was againput over the same column, to ensure that there was no glycatedhemoglobin remaining. Once eluted, this first fraction and the secondfraction are individually dialyzed in a large volume of GG buffer andthen re-concentrated with polyethylene glycol in GG buffer external tothe dialysis bag. This procedure provided a solution of 0%glycohemoglobin (Hb) and a solution of 100% glycohemoglobin (GHb) in GGbuffer.

Alkaline phosphatase conjugated with aminophenyl boronic acid (AP-Bor)was supplied by R & D Systems, Minneapolis, Minn. The stock solution ofAP-Bor was made up as 0.05 mg/mL in GG buffer with magnesium chloride.

In the model assay, the GHb and Hb fractions are first made up to equaloptical density solutions by diluting each by a factor of approximately20 times, and further diluting the Hb fraction to adjust to the OD ofthe GHb fraction. These equal OD solutions were then mixed inproportions to provide GHb to total hemoglobin of 0, 5, 15, 33, 50, 66,7and 100%. In a parallel experiment frozen pooled patient sample withHb/GHb values established by the supplier (Primus Corporation, KansasCity, Mo.), were used in a similar way. These samples are with known GHbof 6.4, 7.5, 10, 12, 13.7, and 18.9%, which covers the clinical expectedrange.

When 50 μL of each analyte sample described in the previous paragraphwas placed in individual wells of a 96 well plate, the optical densityat 540-694 nm is approximately 0.15 OD. This first well content is thenserially double diluted with buffer, and the useful dilutions are thefirst 4 or five as discovered by preliminary experiment. The opticaldensity of all dilutions was recorded before further treatment. This 96well plate was kept on a cold surface until the developer is added.

A solution of AP-Bor was further diluted from stock, by adding 25 μL ofstock to 10 mL of GG buffer and mixing. This solution was maintained inan ice bath until used. 100 μL of dilute AP-Bor was added to each wellof the 96 well plate. Incubation was for 40 minutes.

Developer solution was composed of 11 mL of GG buffer to which is added1 mL of 50 mM acetoacetamide, 300 μL of 3% hydrogen peroxide, and 300 μLof each of 50 mM naphthyl phosphate andN′-ethyl-N′ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine.This was also made up fresh for each assay and kept cold until used. 100μL was added to each well after the incubation is complete. The platewas transferred to a plate reader and optical density was monitored at 5minute intervals for thirty minutes at 694-490 nm. Results of the assaywith purified GHb and Hb are shown in Table 2. Results of the assay withfrozen pooled human samples supplied by Primus Corporation are shown inTable 3. It can be seen that the assay satisfies the requirements forlinearity, which provide evidence for both accuracy and reproducibility.

TABLE 2 OD 694-490 nm of GHb diluted with Hb Developed as in example 5.Normalized to 0 GHb GHb Concentration 4 μL 3 μL 2 μL 1 μL 0.5 μL 0.25 μL0.00 0 0 0 0 0 0 33.33 281 174 80 57 44 35 50.00 392 252 132 100 62 5666.67 494 341 172 109 73 63 100.00 691 486 257 164 116 89 15.00 127 6825 −8 −7 12 5.00 45 36 65 64 66 64 slope 6.948185 0.700435 0.4921320.664246 0.685773 0.717647 intercept 21.99859 −9.26896 9.02533 0.062262.959212 9.278984 r 0.99657 0.997572 0.977143 0.971103 0.976559 0.978467

TABLE 3 Glycated hemoglobin % determined by solution assay using frozenpooled patient samples. Expected Actual 6.4 4.27905 7.5 7.319253 1010.9135 12 11.18437 13.7 13.06465 18.9 19.1984 Slope 1.099554 Intercept−1.56004 r 0.982899

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent or patent application were specifically andindividually indicated to be so incorporated by reference.

I claim:
 1. A method for detecting an analyte in solution comprising a)combining i) a solution to be assayed for the presence or amount of theanalyte; ii) a first ligand that binds the analyte, wherein said firstligand is directly or indirectly bound to a hydrolase that cleaves afirst substrate to produce a colorless first product; iii) a secondligand that binds the analyte, wherein the second ligand is directly orindirectly bound to an oxidase that oxidises a second substrate toproduce a colorless second product; iv) said first substrate; v) saidsecond substrate; vi) a compound that is a scavenger for the firstproduct or the second product in step (a); and, whereby the hydrolasecleaves the first substrate to produce the first product and the oxidaseoxidizes the second substrate to produce the second product, wherein thefirst product and the second product chemically combine, forming acovalent chemical linkage between the first product and second product,to produce a detectable reaction product, said detectable reactionproduct being a colored reaction product; b) detecting the production ofthe colored reaction product; c) relating the production of the coloredreaction product with the presence or amount of analyte in the solution.2. The method of claim 1 wherein the binding of the first ligand to theanalyte does not interfere with the binding of the second ligand.
 3. Themethod of claim 1 wherein the scavenger is3-amino-1-(2,4,6-trichlorophenyl)-2-pyrazolin-5-one or acetoacetamide.4. The method of claim 1 wherein the first substrate is a compound thatcomprises a benzene ring or naphthalene structure with one activehydroxyl group.
 5. The method of claim 4 wherein the first substrate is1-naphthol phosphate or phenyl phosphate.
 6. The method of claim 1wherein the second substrate is selected from the group consisting ofN,N-dimethyl paraphenylene diamine; N,N-diethyl paraphenylene diamine;N-phenyl paraphenylene diamine;N′-ethyl-N′ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine; 4 amino antipyrene; and N,N-dimethylamino benzidine.
 7. Themethod of claim 1 wherein the first ligand is a first antibody thatspecifically binds the analyte and second ligand is a second antibodythat specifically binds the analyte.
 8. The method of claim 1 whereinthe hydrolase is selected from the group consisting of a phosphatase, anesterase, a galactosidase, a lipase, a glucuronidase, an amidase, apeptidase, and a sulphatase.
 9. The method of claim 1 wherein thehydrolase is alkaline phosphatase and the oxidase is horseradishperoxidase.
 10. The method of claim 9 wherein the first substrate isnaphthyl phosphate or phenyl phosphate and the second substrate isN′-ethyl-N′ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine.
 11. The method of claim 1 wherein at least one ofthe first and second ligand is an antibody or a lectin.
 12. The methodof claim 7 wherein the hydrolase is selected from the group consistingof a phosphatase, an esterase, a galactosidase, a lipase, aglucuronidase, an amidase, a peptidase, and a sulphatase.
 13. The methodof claim 12 wherein the hydrolase is a phosphatase and the oxidase is aperoxidase.
 14. The method of claim 13 wherein the hydrolase is alkalinephosphatase and the oxidase is horseradish peroxidase.
 15. The method ofclaim 12 wherein the first substrate is 1-naphthol phosphate or phenylphosphate.
 16. The method of claim 12 wherein the second substrate isselected from the group consisting of N,N-dimethyl paraphenylenediamine; N,N-diethyl paraphenylene diamine; N-phenyl paraphenylenediamine;N′-ethyl-N′ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine; 4 amino antipyrene; and N,N-dimethylamino benzidine.
 17. Themethod of claim 12 wherein the first substrate is naphthyl phosphate orphenyl phosphate and the second substrate isN′-ethyl-N′ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine.18. The method of claim 12 wherein the scavenger is3-amino-1-(2,4,6-trichlorophenyl) -2-pyrazolin-5-one or acetoacetamide.19. The method of claim 3 wherein the scavenger is3-amino-1-(2,4,6-trichlorophenyl) -2-pyrazolin-5-one.
 20. The method ofclaim 3 wherein the scavenger is acetoacetamide.
 21. The method of claim3 wherein: a) the hydrolase is alkaline phosphatase and the oxidase ishorseradish peroxidase; and b) the first substrate is naphthyl phosphateor phenyl phosphate and the second substrate isN′-ethyl-N′ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine.
 22. The method of claim 7 wherein: a) the hydrolaseis alkaline phosphatase and the oxidase is horseradish peroxidase; b)the first substrate is naphthyl phosphate or phenyl phosphate and thesecond substrate isN′-ethyl-N′ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine;c) the scavenger is 3-amino-1-(2,4,6-trichlorophenyl)-2-pyrazolin-5-oneor acetoacetamide.